This invention relates to a process for measuring an endotoxin in a sample and an apparatus used therefor.
Endotoxins are a typical fever-producing substance (pyrogen). When a blood, transfusion solution or parenteral solution contaminated by an endotoxin is inserted into a living body, it is known to produce serious side effects such as a high fever or shock. Therefore, it is absolutely necessary to measure the amount of exdotoxin in a raw material solution, washing water, etc., in order to prevent the contamination with the endotoxin in the production steps of medical products such as injections. Further, the measurement of endotoxin is widely applied, for example, to a functional test for ultrapure water producing membranes, or to an examination of washing water used in the production of semiconductors.
Recently, there has been developed a method for measuring an endotoxin by applying a gelation reaction between the Limulus amoebocyte lysate (a component extracted from amoebocytes of Limulus polyphemus, hereinafter referred to as "LAL") and an endotoxin [J. Levin & F. B. Bang, Thromb. Diath. Haemorrh., 19, 186 (1968)]. This method comprises mixing a sample solution and an LAL reagent in a test tube, allowing to stand at a constant temperature for a certain period of time, observing with the naked eye the formation of gel by declining or inverting the test tube, comparing with the results of similar reaction between an endotoxin-containing sample with known concentration and the LAL reagent, and judging semiquantitatively whether the sample solution is endotoxin-positive (+) or negative (-). But since this method requires much skill and the judging standard of positive or negative depends on observer's subjectivity, there are various disadvantages in that the judgement reflects greatly personal differences, the lower limit of judgement is about 0.05 ng/ml and a further lower concentration of endotoxin cannot be detected, and the like. In addition, the strength of gel state in the gelation reaction is so weak that the gel state is easily broken sometimes by a slight swing of the test tube by a water stream in a water recycling type constant temperature bath which is used for maintaining the reaction at a constant temperature such as at 37.degree. C., or by a slight mcve of the test tube when it is taken out of the constant temperature bath for the observation by the naked eye, which results in producing an error. Therefore, it is very difficult to obtain precise measured values stably by this method.
Recently, there is proposed a method for determining the endotoxin content by measuring optically the turbidity of samples and using the changes of absorbances, noticing that the turbidity is produced by the gelation (e.g. Japanese patent unexamined publication Nos. 9050/83 and 42451/84). According to this method, objectivity of the judgement is improved, but since it requires several tens minutes to about 1 hour for the gelation reaction, a longer time is necessary for measuring a plurality of samples and the measuring efficiency is remarkably bad. Further, when a sample moving system is employed in an absorbance measuring apparatus for measuring a large number of samples continuously in order to improve the efficiency, there is a danger of breaking the gel state of samples in the course of gelation due to the weakness of gel as mentioned above. Therefore, it is difficult to say that this method is a practical one for measuring a plurality of samples in a short time.
On the other hand, there is developed a method for determining an endotoxin content by using a chromogenic peptide derivative as a synthetic substrate, conducting a color development by hydrolysis of the substrate with LAL and endotoxin in a sample, and colorimetrically determining the degree of coloring [S. Iwanaga et al., Haemostasis, 7, 183 (1978)]. But this method is not suitable for daily examinations since the measurable range is as narrow as from several pg/ml to 100 pg/ml and the measuring procedures are remarkably complicated.